Project summary Epilepsy is the most common neurological disorder and has associated costs >$15 billion in the US annually (CDC). We focus on developmental cortical malformations (DCMs), a group of pediatric epilepsy syndromes (80% of patients with DCMs have epilepsy) characterized by reorganization of cortical structure. DCMs include polymicrogyria, focal cortical dysplasia, and schizencephaly and are caused by early life insults and genetic mutation. DCMs are the most common cause of pediatric refractory epilepsy (76% of DCM patients do not respond to treatment) and often require surgical intervention. Due to diffuse regions of hyperexcitability, even surgical resection is of limited usefulness. DCMs demand new treatment options. We believe that DCMs arise from disrupted glutamate signaling during early development. The precise timing of glutamate signaling during development is essential to network maturation, therefore, disruptions may have significant long-term consequences. Our studies using the neonatal freeze-lesion (FL) model of DCM demonstrate that early life cortical insult disrupts glutamate levels in the developing cortex. We hypothesize that this alters interneuron activation, disrupting their maturation and later functional properties. Intriguingly, pharmacologically blocking a subtype of NMDA receptors mimics multiple FL-associated pathologies and alters interneuron maturation. Taken together, we hypothesize that disruptions in ambient glutamate during early postnatal development alter interneuron activity at a critical time, lead to disruption of inhibitory networks, and cause lasting cortical network hyperexcitability. To address our hypothesis we will utilize a combination of whole-cell electrophysiological recording, glutamate biosensor imaging, extracellular field recording of network activity, EEG analysis of cortical hyperexcitability, and analysis of inhibitory interneurons. We will ask whether 1) ambient glutamate affects interneuron maturation, 2) FL alters ambient glutamate and leads to changes in IN maturation, and 3) whether altering interneuron activity during early neonatal development (using pharmacological and chemogenetic approaches) disrupts interneuron maturation and induces network hyperexcitability. At the completion of these studies, we will know if disruption interneuron activity during development leads to long term inhibitory hypofunction. We will be poised to engage 2C/D-NMDARs as a preclinical target to improve clinical outcomes for individuals with epilepsy due to DCMs. Additionally, our studies will provide new insight into the relationship between glutamatergic activity, astrocytes, and interneurons in the developing cerebral cortex.